Progression through the cell cycle requires exquisite coordination between DNA replication initiation and the multiple events that must take place in a timely and spatially-specific manner. Proteins involved in replication initiation are remarkably similar in eukaryotes and prokaryotes. Decades of research revealed in fine detail how these proteins function to initiate replication. However, it is becoming increasingly apparent that these proteins also mediate non-replication functions. Little is known about how these functions are coordinated with chromosome replication. The goal of this proposal is to define the mechanisms that integrate multiple cell cycle functions in Caulobacter crescentus, specifically focusing on the bacterial protein responsible for replication initiation, DnaA. Caulobacter is an ideal model for studying bacterial cell cycle regulatory circuitry due to the simultaneous replication and polar segregation of its single chromosome and its asymmetric cell division. Recently, a dnaA mutant that disrupts the coordination of chromosome replication and spatial/temporal control of cytokinesis was identified. The specific aims of this proposal are to determine the role of DnaA in mediating (a) nucleoid occlusion at the incipient division site and (b) the coordination of chromosomal segregation with division site selection using high-resolution imaging combined with engineered strains with specific fluorescent tags and key cell division protein depletions.